This paper combines the topographic imaging capability of the atomic force microscope (AFM) with a compositionally patterned array of immobilized antigenic rabbit IgG on gold as an approach to performing immunoassays. The substrates are composed of micrometer-sized domains of IgG that are covalently linked to a photolithographically patterned array of a monolayer-based coupling agent. The immobilized coupling agent, which is prepared by the chemisorption of dithiobis(succinimidyl undecanoate) on gold, is separated by micrometer-sized grids of a monolayer formed from octadecanethiol (ODT). The strong hydrophobicity of the ODT adlayer, combined with the addition of the surfactant Tween 80 to the buffer solution that is used in forming the antibody−antigen pairs, minimizes the nonspecific adsorption of proteinaceous materials to the grid regions. This minimization allows the grids to function as a reference plane for the AFM detection of the height increase when a complementary antibody−antigen pair is formed. The advantageous features of this strategy, which include ease of sample preparation, an internal reference plane for the detection of topographic changes, and the potential for regeneration and reuse, are demonstrated using rabbit IgG as an immobilized antigen and goat anti-rabbit IgG as the complementary antibody. The prospects for further miniaturization are discussed.